The present invention relates to the field of micromechanical devices as sensors for detecting physical or chemical changes caused by chemical interactions between naturally occurring bio-polymers which are non-identical binding partners, such as can occur with polyamino acids, polynucleotides, and the like. The method of the present invention is useful whether the reactions occur through electrostatic forces or through other forces. In particular, the present invention provides a method for detecting chemical interactions between naturally occurring bio-polymers which are non-identical binding partners where one binding partner or probe molecule is placed on a cantilever for possible reaction with a sample analyte molecule (i.e., a non-identical binding partner). The physical or chemical change may be induced stress, heat, or mass, for example. The present invention is particularly useful in determining DNA hybridization but may be used in detecting interactions between any analyte molecules, whether monomeric or polymeric. Examples of polymer arrays which can be used with the method of the present invention include nucleic acid arrays, protein or polypeptide arrays, carbohydrate arrays, and the like.
As known in the art, various techniques have been used to determine whether a chemical interaction has occurred between two materials, such as between a probe carrying a binding partner and a sample. In the specific example of determining whether DNA hybridization has occurred, various techniques have been used to extract information from a sample. For example, detection schemes have been used that are responsive to fluorescence in order to reveal specific interactions or hybridizations. U.S. Pat. No. 5,578,832, xe2x80x9cMethod and Apparatus for Imaging a Sample on a Device,xe2x80x9d issued to Trulson et al. (xe2x80x9cthe ""832 patentxe2x80x9d) and U.S. Pat. No. 5,631,734, xe2x80x9cMethod and Apparatus for Detection of Fluorescently Labeled Materials,xe2x80x9d issued to Stern et al. (xe2x80x9cthe ""734 patentxe2x80x9d) provide methods and systems for detecting a labeled marker on a sample located on a support through the use of an excitation radiation source and radiation optics. The ""832 patent and the ""734 patent are hereby incorporated by reference for all they disclose and for all purposes. As described in the ""832 and ""734 patents, these techniques employ the use of a label, for example, the DNA probe is labeled with a fluorescent molecule, such as fluorophore or biotin. Once the DNA probe is labeled according to prior methods, an optical system can be used to determine whether hybridization has occurred by measuring fluorescence activated between the labeled sample and the probe material.
The present invention provides a method for determining whether a chemical interaction has occurred between naturally occurring bio-polymers which are non-identical binding partners through detecting a physical or chemical change on a micromechanical device called a cantilever. A cantilever, by way of analogy, can be thought of as a diving board which has been reduced to a very small size. More specifically, a cantilever is a physical device that is attached to another object at one end and remains free to move on the other end. Deflection or up and down movement of the free end of the cantilever can then be detected. The method of the present invention can be used with any chemical analyte to generate a physical or chemical change, whether through affinity binding, which may include hydrogen bonding, electrostatic attractions, hydrophobic effects, dipole interactions, or through other forces.
The use of micromechanical sensors is advantageous in the method of the present invention for several reasons. Various signals such as force, heat, stress, magnetism, charge, radiation and chemical reactions can be readily transduced into a micromechanical deflection by an appropriately coated structure, such as a cantilever. In addition, silicon-based micromechanical devices can easily be integrated into microelectronic processing systems such as CMOS (Complementary Metal-Oxide-Semiconductor), as known to one of skill in the art. As a result, it is possible to produce seamless sensors as low cost and to integrate them directly into computers. Moreover, micromechanical sensors are very small, typically approximately 400 xcexcm in length, approximately 40 xcexcm wide and approximately 1 xcexcm thick. As a result, it is possible to obtain a short response time, generally measured in microseconds, as well as sensitivity superior to standard techniques. Finally, it is possible to construct arrays of micromechanical devices, thereby permitting complex analysis of a variety of signals as well as the use of a variety of sensing materials.
By way of background, it is known that stress induced by self-assembled monolayers can be detected by observing the deflection of a micromachined cantilever similar to those used in the commercial Atomic Force Microscope (xe2x80x9cAFMxe2x80x9d), as described by Berger et al., in xe2x80x9cSurface Stress in the Self-Assembly of Alkanethiols on Gold,xe2x80x9d Science, Jun. 27, 1997, Vol. 176, p. 2021 (xe2x80x9cBerger Ixe2x80x9d), which is hereby incorporated by reference for all it teaches. The Berger et al. paper studied the surface stress changes during self-assembly of selected molecules, including alkanethiol molecules self-assembled on gold. The researchers found that the stress increases linearly with the length of the alkyl chain of the molecule. In addition, the researchers detected a change in the state of stress with the formation of salt bridges formed when mercaptohexadecanoic acid was deposited on a functionalized surface coated with the self-assembled thiols. This change in cantilever stress was used to detect the formation of the salt bridges when the analyte molecules were introduced.
Other pertinent work involving michromechanical sensors is reflected in a paper by Berger et al. entitled xe2x80x9cNanometers, Picowatts, Femtojoules: Thermal Analysis and Optical Spectroscopy Using Micromechanics,xe2x80x9d Analytical Methods and Instrumentation, Special Issue, xcexcTAS ""96 (xe2x80x9cBerger IIxe2x80x9d), also incorporated by reference for all it discloses and for all purposes. In Berger II, examples of low-cost, disposable micromechanical devices are described which perform optical absorption spectra, calorimetric and thermal analysis, electrochemical stressograms, gas phase adsorption and surface reaction monitors.
Other work in the area of micromechanical sensors is reported by Gimzewski et al. in xe2x80x9cObservation of a chemical reaction using a micromechanical sensor,xe2x80x9d Chemical Physics Letters, Vol. 217, No. 5,6, Jan. 28, 1994, (xe2x80x9cGimzewskixe2x80x9d) which is hereby incorporated by reference for all it discloses and for all purposes. Gimzewski discloses a calorimeter for sensing chemical reactions. The device is based on a micromechanical silicon lever coated with a layer of aluminum. A sample is deposited on the lever in a thin layer. Heat fluxes are detected by measuring the deflection of the cantilever induced by the differential thermal expansion of the lever. Specifically, Gimzewski discloses using this technique to review the catalytic conversion of H2+O2 to obtain H2O.
It is further known to operate multiple probes for the atomic force microscope. As described by Minne et al., xe2x80x9cAutomated parallel high-speed atomic force microscopy,xe2x80x9d Applied Physics Letters, Volume 78, No. 18, May 4, 1998 (xe2x80x9cMinnexe2x80x9d), which is herein incorporated by reference for all it discloses and for all purposes, an expandable system is provided to operate multiple probes for the atomic force microscope in parallel at high speeds. The cantilever footprint is only 200 xcexcm wide which allows the devices to be placed in a one-dimensional expandable parallel array.
Yet another contribution to the art of micromechanical sensors is described by Manalis et al., xe2x80x9cInterdigital cantilevers for atomic force microscopy,xe2x80x9d Applied Physics Letters, Vol. 69, No. 25, Dec. 16, 1996 (xe2x80x9cManalis Ixe2x80x9d), which is hereby incorporated by reference for all it discloses and for all purposes. In Manalis I, an AFM sensor is described in which a silicon cantilever is micromachined into the shape of interdigitated fingers that form a diffraction grating. When detecting a force, alternating fingers are displaced while remaining fingers are held fixed. As a result, a phase sensitive diffraction grating is created which allows the cantilever displacement to be determined by measuring the intensity of diffracted modes.
Another paper by Lang et al., xe2x80x9cSequential position readout from arrays of micromechanical cantilever sensors,xe2x80x9d Applied Physics Letters, Vol. 73, p. 383, 1998 (xe2x80x9cLangxe2x80x9d) describes using a reference cantilever for canceling environmental noise. Lang is hereby incorporated by reference for all it discloses and for all purposes. In Lang, chemically specific responses are extracted in a noisy environment using a sensor to detect specific chemical interactions and an uncoated cantilever as a reference.
Finally, another paper by Manalis et al., xe2x80x9cTwo dimensional micromechanical bimorph arrays for detection of thermal radiation,xe2x80x9d Applied Physics Letters, Jun. 16, 1997, (Manalis II) hereby incorporated by reference for all it discloses and for all purposes, describes fabricating arrays of cantilevers and using them as sensitive detectors of head induced stress. Specifically, the cantilevers described by Manalis II were placed on a grid with 50 microns on centers. The present inventors have determined that this type of array is a suitable substrate for determining, for example, hybridization.
Rather than using traditional labeling, such as optical or electrochemical labeling, in order to detect chemical interactions between naturally occurring bio-polymers which are non-identical binding partners, the present inventors have determined a new and useful method for xe2x80x9creadingxe2x80x9d a substrate to determine whether a particular chemical interaction has occurred. In traditional labeling, sample analyte molecules are modified in some way to permit their detection when they combine with the probe molecules. The method of the present invention is particularly useful in the detection of hybridized sites on a DNA probe array. The method of the present invention allows detection of hybridization without modifying either the analyte or the probe molecules, i.e., it requires no labeling.
According to the method of the present invention, a chemical interaction between naturally occurring bio-polymers which are non-identical binding partners is monitored by detecting a physical or chemical change through deflection of a cantilever. The physical or chemical change can be, for example, induced stress on the cantilever which causes the cantilever to move or deflect. Standard AFM techniques are then used to detect the deflection of the cantilever. The physical or chemical change can also be in the form of a heat reaction, which similarly causes the cantilever to deflect or bend where the cantilever is made of two materials, i.e., is a bimorph. A physical or chemical change might also result in a change in mass on the cantilever. In such an example, the resonant frequency of the cantilever will change due to the mass change. Measuring the resonant frequency of the cantilever under such circumstances will allow the physical or chemical change to be detected.
In a specific embodiment of the present invention, oligonucleotides are deposited onto a cantilever. The stress induced by hybridization is detected with methods commonly used for detecting cantilever deflection in the AFM. As is well known to one skilled in the art, these methods are sensitive to the point where deflections less than 0.01 nm can be easily detected. The substrate used according to the method of the present invention allows exploitation of the cantilever""s properties in order to detect the hybridized sites.
Specifically, the stress in the individual cantilevers is monitored in the manner shown by Manalis II, noted above. First, the surfaces of the cantilevers are prepared in the same manner now common in immobilized sensor technology, as known to one skilled in the art. Next, a binding partner, such as oligonucleotides, is deposited on the cantilevers to form an array of probes. This deposit will change the state of stress on the individual cantilevers and this stress pattern is used as the reference. When sample or analyte molecules (i.e., a non-identical binding partner) are introduced to the cantilever and interact with the binding partner (probe molecules) at appropriate sites, the stress on the cantilever at the particular site will change as a result of the interaction. The change in stress with the introduction of the sample molecules will be monitored with standard AFM techniques.
The present invention does not use optical or electromechanical labels, as previously described. In addition, it serves as a tool for understanding the processes involved in chemical interactions between naturally occurring bio-polymers which are non-identical binding partners, such as DNA hybridization, by providing additional ways to measure events such as the length of the chemical interaction and the number of molecules hybridized. Moreover, it provides an additional, highly sensitive, low-cost means to monitor chemical interactions, as described in detail below.